The classical techniques for isolating plasmid DNA from microbial fermentations are suitable for small or laboratory scale plasmid preparations. One such procedure involves the alkaline lysis of microbial host cells containing the plasmid, followed by acetate neutralization causing the precipitation of host cell genomic DNA and proteins which are then removed by, for example, centrifugation. The liquid phase contains the plasmid DNA which is alcohol precipitated and then subjected to isopycnic centrifugation using CsCl in the presence of ethidium bromide. The ethidium bromide is required in order to separate the total plasmid DNA into the three different forms, supercoiled (form I), nicked circle (form II), and linearized (form III), and the desired plasrnid form is collected. Further extraction with butanol is required to remove residual ethidium bromide followed by DNA precipitation using alcohol. Additional purification steps follow to remove host cell proteins. The removal of host proteins is performed by repeated extractions using phenol or a mixture of phenol and chloroform. The plasmid DNA is alcohol precipitated and residual phenol is removed by repeated isoamyl/chloroform extractions. The final alcohol precipitated plasmid DNA is dissolved in water or a suitable buffer solution.
There are numerous drawbacks and limitations to this process including:
a) this process requires the use of expensive and hazardous chemicals (CsCl and EtBr, which are used within the density gradient centrifugation; EtBr is a known mutagen and must be removed from products; also it is an intercalating agent which can nick the plasmid); PA1 b) the density centrifugation step is not easily scaleable; PA1 c) there is a need for organic solvent extraction to remove residual EtBr; PA1 d) phenol extraction is used to remove residual proteins and DNase, a process that would require a centrifuge to break phenol/water emulsion; PA1 e) highly repetitive steps making it laborious and time consuming (isolation requires several days); PA1 f) scalability of the chemical lysis step is an obstacle i.e., lysozyme/alkaline/KOAc treatment step is efficient in lysing cells on a small scale, however, the increase in viscosity makes large scale processing very difficult; and PA1 g) use of large quantities of lysozyme to enzymatically weaken the microbial cell wall prior to lysis. PA1 a) size exclusion chromatography, which is inherently limited in throughput; PA1 b) hydroxyapatite chromatography, which has the disadvantage of requiring high concentrations of urea for efficiency; PA1 c) reversed phase chromatography; and PA1 d) ion exchange chromatography.
The mixture is then neutralized by addition of acid which results in precipitation of the high molecular weight chromosomal DNA. The high molecular weight RNA and protein-SDS complexes precipitate with the addition of high concentration of KOAc salt. The plasmid product remains in the clarified supernatant following centrifugation. Limitations here include the need to process quickly and on ice in order to retard the activity of nucleases which are not removed until phenol extraction. The main contaminant remaining in the supernatant with the product is RNA.
Another commonly utilized method for isolating and purifying plasmid DNA from bacteria provides a rapid process suitable for only very small scale preparations.
Holmes and Quigley (1981, Analytical Biochem., 114, pp 193-197) reported a simple and rapid method for preparing plasmids where the bacteria are treated with lysozyme, then boiled at about 100.degree. C. in an appropriate buffer (STET) for 20-40 seconds forming an insoluble clot of genomic DNA, protein and debris leaving the plasmid in solution with RNA as the main contaminant. Lysozyme is apparently a requirement for this technique to work, and as such adds a treatment step which is less desirable for large scale manufacture of DNA for human or veterinary use. However, the addition of lysozyme may enhance plasmid release during lysis. An advantage is that heat treatment of the cells would also denature the DNase. However, this technique is not suitable for scale up to a high volume of microbial fermentations and is meant for fermentations less than five liters.
Alternatives to isopycnic centrifugation using CsCl for plasmid purification have been published. These alternatives are suitable only for laboratory scale plasmid isolation and include:
Large scale isolation and purification of plasmid DNA from large volume microbial fermentations, therefore, requires the development of an improved plasmid preparation process. An isolation and purification process for large scale plasmid DNA production is necessitated by recent developments in many areas of molecular biology. In particular, recent advances in the field of polynucleotide-based vaccines for human use, and potentially human gene therapy, requires the ability to produce large quantities of the polynucleotide vaccine in a highly purified form.
Unprecedented technology is required for developing/-implementing a large scale commercially viable process for fermentation, isolation, purification and characterization of DNA as a biopharmaceutical.